Antisense glutaminase inhibition decreases glutathione antioxidant capacity and increases apoptosis in Ehrlich ascitic tumour cells Jorge Lora, Francisco J. Alonso, Juan A. Segura, Carolina Lobo, Javier Ma ´ rquez and Jose ´ M. Mate ´ s Departamento de Biologı ´ a Molecular y Bioquı ´ mica, Laboratorio de Quı ´ mica de Proteı ´ nas, Facultad de Ciencias, Universidad de Ma ´ laga, Spain Glutamine is an essential amino acid in cancer cells and is required for the growth o f many o ther cell t ypes. Glutami- nase activity is positively correlated with malignancy in tumours and with growth rate in normal cells. In the present work, Ehrlich ascites tumour cells, and their derivative, 0.28AS-2 cells, expressing antisense glutaminase mRNA, were assayed for apoptosis induced by methotrexate and hydrogen peroxide. I t is s hown t hat E hrlich a scites tu mour cells, expressing antisense mRNA for glutaminase, contain lower levels of glutathio ne than normal ascites cells. In addition, 0.28AS-2 cells contain a higher number of apop- totic cells and are more sensitive to both methotrexate and hydrogen p eroxide toxicity than normal cells. T aken together, these results provide insights into the role of glutaminase i n a poptosis by demonstrating that t he expres- sion of antisense mRNA for glutaminase alters apoptosis and g lutathione antioxida nt c apacity. Keywords: antisense; apoptosis; glutaminase; glutamine; glutathione. Phosphate-activated glutaminase (GA, EC 3.5.1.2) has a critical role in tumours and rapidly dividing cells, and its activity is correlated with malignancy [1]. Ehrlic h ascites tumour cells (EATC), transfected with the pcDNA3 vector containing an antisense s egment (0.28 kb) of rat k idney GA, showed impairment in the rate of growth and a reduction in the GA protein level, when compared with the parental cells. Cells were selected after culture for 2–3 weeks. Following G418 selection, 12 drug-resistant (neo + ) clones were picked randomly from different plates and studied after expansion [2]. The transfected cells, named 0.28AS-2, displayed remarkable changes in their morphology and interestingly had lost their t umorigenic capacity in vivo [2]. Glutamine is one of the precursor amino acids i n the biosynthesis of glutathione (GSH) [3]. In a ddition, gluta- mine is a source of glutamate in many locations, and has been shown to p reserve total GSH levels after oxidative damage [4]. GSH (c-glutamyl-cysteinyl-glycine) is the most abundant low-molecular-mass thiol, and GSH/ oxidized glutathione (GSSG) is the major redox couple that determines the antioxidative capacity of cells [5 ]. GSH plays important roles in antioxidant defense, and in the regulation of cellular events such as cell proliferation and apoptosis [6]. On the other hand, its deficiency contributes to oxidative s tress [ 7], w hich plays a key role i n t he pathogenesis of many diseases, including cancer [8]. Glutamine is p articularly associated with increased proliferation and decreased apoptosis in intestinal epith elial cells [9,10] and w hite blood cells [11]. Augmentation of cell apoptosis and i nhibition of tumour growth by glutamine depletion seems to be associated with decreased antioxida- tive GSH-dependent activity and its requirement during cell proliferation [12]. Recent findings support the fact that that the extracellular glutamine level affects the susceptibility of cells to different apoptosis triggers. In fact, glutamine- starving cells contain a reduced level of the antioxidant GSH [13]. 0.28AS-2 cells have been used in this study as a model with reduced GA ac tivity in comparison to the parental EATC. We have characterized the effect of GA inhibition on both GSH-dependent antioxidant capacity and apopt- osis. The cellular redox potential and the intrac ellular GSH : GSSG ratio was measured in this study and the possible i mplications of their d ifferent le vels in cells expressing high or low levels of GA will be discussed. Different inducers of apoptosis [methotrexate (MTX) and H 2 O 2 ) have been used to facilitate a better understanding of the molecular basis of the ir toxicity in r elation to GS H levels and GA inhibition. In fact, MTX, a structural analogue of folic acid, i s widely used in antimetabolite c ancer therapy, demonstrating consistent activity against several malignant tumours [14]. Another a spect of this work is the discovery of new insights into the role of MTX in apoptosis. To illustrate this point, annexin V–fluorescein isothiocyanate (FITC) assays, caspase-3 activity and DNA ladder experiments were carried out with and without MTX. These results, discussed in more detail below, suggest that the use of Correspondence to J. M. Mate ´ s, Department Biologı ´ aMoleculary Bioquı ´ mica, Facultad de Ciencias, Campus de Teatinos, Universidad de Ma ´ laga, 29071 Ma ´ laga, Spain. Fax: +34 952 132000, Tel.: +34 952 133430, E-mail: jmates@uma.es Abbreviations: EATC, Ehrlich ascitic tumour cells; DCF, 2¢,7¢- dichlorofluorescein; DCFH-DA, 2¢,7¢-dichlorodihydrofluorescein diacetate; FITC, fluorescein isothiocyanate; FSC, forward scatter; GA, phosphate-activated glutaminase; GR, glutathione reductase; GSH, glutathione; GSSG, oxidized glutathione; MTT, 3-[4,5-di- methylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; MTX, methotrexate; PI, propidium iodide. Enzymes: phosphate-activated glutaminase (EC 3.5.1.2); glutathione reductase (EC 1.6.4.2). (Received 11 June 2004, revised 13 S eptember 2004, accepted 20 September 2004) Eur. J. Biochem. 271, 4298–4306 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04370.x chemotherapeutic agents, in combination with GA inhibitors, should be taken into account when design ing treatment strategies for cancer. Materials and methods Cell lines EATC (ATCC, Manassas, VA, USA) and i ts derivative, 0.28AS-2, were grown in RPMI medium (Sigma) sup- plemented w ith 10% F BS, 100 units ÆmL )1 penicillin, 100 mgÆmL )1 streptomycin and 1.25 mgÆmL )1 amphoteri- cin (BioWhittaker, W alkersville, MD, USA). Cultures were incubated in a humidified atmosphere, in 5% CO 2 /95% air, at 37 °C. 0.28AS-2 cells were obtained b y EATC lipo- fection, using the lipid Dosper (Boehringer Mannheim, Mannheim, Germany), with the plasmid pcDNA3 contain- ing a n antisense 3¢ cDNA segment (0.28 kb) of rat kidney GA [2]. Assessment of cell growth and viability Cells were enumerated by using a haemocytometer and a ZM Coulter Counter (Coulter, Luton, UK). Prior to apoptosis evaluations, i nhibitory dose 50% (IC 50 )values were determined in clonogenic survival assays of EATC and 0.28AS-2 cells at various concentrations of MTX and H 2 O 2 . In order to characterize the time-course action of concen- tration o f t hese chemicals on cell proliferation, cell viability was examined by using the 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT)/cytotoxicity t est assay. Cells were seeded at a concentration of 2 · 10 4 cellsÆmL )1 in 96-well culture plates. After 24 h of incubation, cells were rinse d with NaCl/P i (PBS) a nd further incubated with fresh medium containing MTX or H 2 O 2 . Finally, c ells were treatedwithMTXorH 2 O 2 at 24, 48, 72 or 96 h, and cell viability was assayed b y using the MTT method. Briefly, MTT (Sigma) was added to the cells at a concentration of 0.5 mgÆmL )1 and the cells were then incubated at 37 °Cina CO 2 incubator f or 3 h. V iable c ells gener ate insoluble crystal, but cells float and are loosely attached to the surface of culture plates. Therefore, to avoid the potential loss of cells, and to dissolve the insoluble crystal generated by the cells, 100 lLof0.04 M HCl in 2-propanol was a dded directly to each well. After 30 min, the sample absorbance was measured at 570 nm by the use of an ELISA microplate reader, and the results were analysed by using SOFTMAX PRO software (Molecular Devices, Sunnyvale, CA, USA). GSH antioxidant system Reduced glutathione (GSH) plus oxidized glutathione (GSSG) levels were determined b y using a m ethod described previously [15]. Approximately 5 · 10 6 cells were disrupted in ice-cold 1 M perchloric acid containing 2 m M EDTA. The homogenate was then centrifuged at 15 000 g for 5 min at 4 °C to obtain the supernatant. An aliquot (0.5 mL) of the acidic supernatant was neutralized with 0.5 mL of 2 M KOH containing 0.3 M Mops. The sample was assayed for GSH and GSSG immediately after neutralization [16]. The reaction mixture comprised 0.26 m M NADPH, 76 l M 5,5¢-dithiobis(2-nitrobenzoic acid) (DTNB) and t he sample, in 0.2 mL. The final volume was adjusted to 0.3 mL with NaCl/P i (0.1 M containing 1 m M EDTA, pH 7 .0) and the absorbance was monitored at 412 nm for 120 s after the addition of glutathione reductase (GR) (0.06 unitsÆmL )1 ). To determine the amount of GSSG in the sample, an identical 0.3 mL neutralized sample was i ncubated f or 60minwith1lL of 4-vinilpyridine, and assayed as described above [17]. GR activity was evaluated by using a method based on that previously described by Carlberg & Mannervik, with minor modifications [18]. We used 3 · 10 6 cells that were washed with ice-cold 200 m M NaCl/P i (pH 7 .0) and frozen at )80 °C. Cells were thawed in 0.5 mL of buffer containing 1m M EDTA, 50 l M phenylmethanesulfonyl fluoride, 0.1 m M dithioerythritol an d 200 m M NaCl/P i (pH 7 .0). The homogenate was refrozen until required for use. The first reaction mixture c omprised 0.1 m M NADPH, 1 m M EDTA, 100 m M NaCl/P i (pH 7.0) and 100 lLofsample in 1 mL of final v olume. In this assay, the oxidation of NAPH, independently of GR, was determined as the decrease of absorbance at 340 nm for 60 s. Finally, we added 50 lLof20m M GSSG, and the decrease in absorbance at 340 nm was monitored for an additional 120 s . One unit of GR activity was defined as 1 lmol of NADPH oxidized per minute. Quantification of reactive oxygen species (ROS) EATC and 0.28AS-2 cells (5 · 10 5 perwell),tobeusedfor ROS quantification, were exposed to 10 l M 2¢,7¢-dichloro- dihydrofluorescein diacetate (DCFH-DA) at 37 °Cfor 10 min . They were then washed twice with NaCl/P i and lysed with 10 m M Tris/HCl buffer, pH 7.4, containing 0.5% Tween-20. The homogenates were centrifuged at 10 000 g for 10 min to remove cell debris. This method is based on the oxidation of DCFH-DA by ROS, resulting in the formation of t he fluorescent c ompound, 2¢,7¢-dichlorofluo- rescein (DCF). D CF fluorescence in the supernatant w as measured by using a spectrofluorometer with excitation of 500 n m and emission by scanning from 500 to 550 nm. GA, glutamine and glutamate assays GA activities were determined by using a previously described method [ 2]. The contents of glutamine and glutamate w ere determined according to the method described by Baverel & Lund, with slight modifications [19]. Blanks with samples o mitted were also run in parallel. Intracellular concentrations of glutamine and glutamate were calculated as previously described [ 20]. DNA ladder assay For DNA ladder assays, 1 · 10 6 cells were harvested and washed with NaCl/P i . Alternatively, cells were collected after i ncubation with 100 n M MTX f or 48 h, or with 100 l M H 2 O 2 for 24 h , and washed with NaCl/P i .Cell pellets were r esuspended in l ysis buffer [100 n M NaCl, 10 m M Tris/HCl, 24 m M EDTA, and 0.5% (v/v) SDS] containing 0.1 mgÆmL )1 proteinase K, and then incubated at 55 °C overnight [21]. DNA was extracted by using an equal volume of phenol/chloroform (v/v) and p recipitated Ó FEBS 2004 Effect of glutaminase on glutathione and apoptosis ( Eur. J. Biochem. 271) 4299 by adding absolute ethanol and 0.3 M ammonium acetate at )20 °C overnight. The DNA was resuspended in sterile water, treated with RNase at 3 7 °C for 1 h, and then analysed by gel electrophoresis on a 1% (w/v) agarose gel containing 0.5 lgÆmL )1 ethidium bromide i n both gel and running buffer. Phosphatidylserine membrane asymmetry assay For the detection of annexin V, 5 · 10 5 cells were incubated with the respective chemicals, harvested, washed with NaCl/ P i and then suspended in binding buffer. Cells were stained with FITC-labelled annexin V, a s described by the manu- facturer (annexin V-FITC assay kit purchased from MBL Co. Ltd, Nagoya, Japan), and 10 lL of a stock s olution o f propidium iodide (PI, 20 lgÆmL )1 ). PI is excluded from viable cells and commonly used f or identifying dead cells as a counterstain in multicolor fluorescent techniques [22]. After staining, cells were maintained on ice until analysis. Flow cytometric determinations were performed by using a FACSort analyzer (Becton-Dickinson, Franklin Lakes, NJ, USA)andanair-cooled,15mWargon-ionlasertunedat 448 n m. For each cell suspension, 10 4 events we re collected and the following parameters were recorded simultaneously for each cell: forward scatter (FSC ), as an estimation of cell size and refractive index, right-angle light scatter (NaCl/ Cit), as an estimation of cell complexity, green fluorescence (FL1, 525 nm) to determine annexin V -FITC binding, and orange (FL2, 5 75 nm) and red ( FL3, 6 60–675 nm) fluores- cence to quantify the incorporation of P I [23]. FSC a nd NaCl/Cit signals were amplified lineally, and FL1, FL2, and FL3 signals were amplifie d logarithmically. All measure- ments were recorded and stored as listmode files. Numerical analysis was performed off-line by using CELLQUEST software (Becton-Dickinson), as described previously [24]. FSC vs. NaCl/Cit dot-plots were used to exclude debris and aggregates from the analysis. Annexin V is detected on the cell membrane i n early p hase apoptotic cells, which is followed by the formation of both annexin V-positive and PI-positive late apoptotic cells. FACS analysis, using anti-annexin V immunoglobulin and P I, revealed the formation of both annexin V-positive and PI-negative early apoptotic cells and annexin V-positive and PI-positive late apoptotic cells [25]. This discrimination is currently in use in flow cytometric analysis, and so early and late apoptotic cells were included in our analysis. Measurement of caspase-3 activity The enzymatic activity of caspase-3 was determined by using the chromogenic s ubstrate DEVD-pNA, containing a specific cleavage site (DEVD) linked to p-nitroanilide (Caspase-3/CPP32 colorimetric assay kit; M BL Co. L td). EATC and 0.28AS-2 untreated cells, or cells treated f or 48 h with 100 n M MTX, were washed twice w ith NaCl/P i , suspended in lysis buffer and then incubated on ice for 10 min after which cell debris was removed by centrifuga- tion and s upernatants were used to determine enzyme activity and p rotein content [26]. Sample protein c oncen- trations were measured by using the Bio-Rad Protein Assay System (Hercules, CA, USA). Samples were normalized for protein concentration, added to a reaction buffer containing 400 l M DEVD-pNA and incubated for 60 min at 37 °C. The p-nitroanilide was quantified by using a spectrophotometer (Shimadzu, Kyoto, Japan) to determine absorbance (A) values a t 405 nm. Calculations were performed to determine the caspase activity (min )1 Ælg )1 of protein) of each sample. Statistical analysis In the MTT assay, and i n experiments carried out to determine GR a ctivity, GSH and GSSG contents and caspase-3 activity, the result of each experiment is expressed as the m ean and SD from at least three independent values. Other data are representative of three individual experi- ments, where s imilar r esults were obtain ed o n e ach occasion. The statistical significance of e xperimental data was eval- uated b y using the Mann–Whitney U-test. A P value of < 0.05 was considered as statistically signific ant. Results Effect of the antisense targeting GA expression on the GSH-dependent antioxidant system and on apoptosis GSH-dependent antioxidant levels, in response t o the inhibition of GA expression, are presented in Table 1. The inhibition of GA expression decreases the level of GSH, resulting in a 40% re duction of the GSH/GSSG r atio. This effect was accompanied by a lower activity of GR (60%) compared w ith the control values of GR activity in E ATC. In addition, cells transfected with random antisense mRNA, which did not affect GA activity, did not show lower GSH levels (results not shown). Therefore, specific inhibition of GA expression using a ntisense technology may result in a reduced or dysfunctional GSH-dependent antioxidant sys- tem. As 0.28AS-2 cells have a decreased GSH antioxidant capacity, they have an increase i n ROS, as shown i n Table 1 . Additionally, a fourfold increase in the c oncentra- tion of glutamine is found in 0.28AS-2 cells compared with EATC (Table 2). Surprisingly, no differences w ere d etected in the intracellular concentrations of glutamate, despite the higher glutamine level detected in 0.28AS-2 cells (Table 2). To evaluate apoptosis, flow cytometric quantification w ith annexin V-FITC w as employed. A smaller p roportion of EATC than of 0.28AS-2 cells were apoptotic. In fact, twice as many apoptotic cells were found in EATC than in 0.28AS-2 Table 1. Effect of antisense mRNA glutaminase expression on the glutathione ( GSH) antioxidant capacity and on the reactive oxygen species (ROS) levels. Results r epresent the mean values ± SD for three different determinations. Assays were performed on individual cell culture plates, and the mean o f duplicate analysis w as used for s tatis- tical calculations. GSSG, o xidized glutathione. Cells GSH (nmolÆg )1 ) GSSG (nmolÆg )1 ) GSH/GSSG ROS (relative units) EATC 4320 ± 1390 33 ± 0.6 131 ± 27 1.00 ± 0.08 0.28AS-2 2670 ± 210* 34 ± 4 78 ± 5* 1.35 ± 0.07* *P< 0.01 comparing control Ehrlich ascitic tumour cell (EATC) values with those of 0.28AS-2 cells. 4300 J. Lora et al. (Eur. J. Biochem. 271) Ó FEBS 2004 cells (Table 3). Consequently, the effect of constitutive apoptosis represents a higher degree o f apoptosis for 0.28AS-2 cells, a s twice as many apoptotic cells are present despite their reduced proliferation rate [2]. On t he other hand, EATC transfected with random antisense m RNA, which did not affect GA activity, did not show a greater tendency for apoptosis than control cells (results not shown). Hence, our model strongly suggests that the inhibition of antisense GA is linked to the activation o f apoptosis. To further confirm the observation that antisense GA expression induces apoptosis, we tested caspase-3 activity in 0.28AS-2 c ells and EATC. This enzyme plays a critical role in the execution of apoptosis and it is r esponsible for many of the biochemical and morphological changes associated with it; consequently, caspase-3 activity has been widely used to diagnose cells undergoing apoptosis [27]. In agreement with this finding, caspase-3 activity was 65% higher in 0.28AS-2 cells compared to the EATC control cell line (Table 3). GR activity in response to MTX action The inhibitory actio n of MTX o n several NAD + (P)- dependent dehydrogenases gave rise to the hypothesis t hat this drug could promote alterations in the redox state [28]. GR is a protein found in both cytosol and mitochondria, whose activity is NADPH dependent [29]. Being also a dehydrogenase, in spite of the decreased l evels of NADPH caused by MTX in p reviously published r eports [30], the activity of GR could b e directly affected by the d rug. In fact, the specific activity of GR present in cells treated with 100 n M MTX was significantly diminished (P <0.01for EATC, and P < 0 .05 f or 0.28AS-2 cells) compared to untreated cells (Table 3). In our study, GR activity of EATC was strongly decreased to l ess than h alf of that f ound when the drug was present. Interestingly, GR activity was only s lightly reduced in 0.28AS-2 cells when 100 n M of GR was present for 48 h (Table 3). As the remaining activity was minimal in both c ases, it is presumed that both c ytosolic and mitochondrial forms of GR are i nhibited by 100 n M MTX. This result reinforces the h ypothesis that MTX interferes with the maintenance of intracellular levels of reduced GSH, suggesting that the cells could be more sensitive to ROS [31]. Dose rate effect of MTX and H 2 O 2 on cell viability In order to a nalyse the biological effect associated with the down-regulation of GA expression, the growth of EATC and 0.28AS-2 cells during treatment with MTX and H 2 O 2 was investigated by using the MTT assay. First, we analysed the growth curve of EATC and 0 .28AS-2 cells at baseline, and at 24, 48, 72 and 96 h, to illustrate the differences in their growth after seeding with similar number of cells. Clones transfected with the 0.28 kb antisense GA cDNA segment showed a decrease in g rowth rate (Fig. 1). Chem- icals were administered to the cells 24 h after plating, and the effects o f MTX and H 2 O 2 were examined after 24, 48, 72 and 96 h of exposure, respectively. As shown by the MTT assay, the l ong-term action of both MTX and H 2 O 2 (up to 96 h) resulted in a dose-dependent loss of viability ( Figs 2 and 3). The concentrations employed were 16, 32, 64, 128, 256, 512, 1024 and 2048 n M . MTX, at c oncentrations higher than 64 n M , s ignificantly i nhibited the proliferation of both EATC and 0.28AS-2 cells after 48 h of exposure (results not shown). This effect was also observed at a lower concen- trations of the drug (16 n M ,32n M and 64 n M )onthe 0.28AS-2 cells. When cells were treated for 96 h, at concentrations lower than 128 n M , there was a larger decrease in the proliferation of 0.28AS-2 cells in comparison to EATC (Fig. 2). The H 2 O 2 concentrations used in this experiment were 0.78, 1.56, 3.13, 6 .25, 12.5, 25, 50 and 100 l M .After96hof incubation with H 2 O 2 , the differences between 0.28AS-2 cells and EATC were significant (0.78, 1.56, 12.5 and 25 l M : P < 0 .01; and 3.13 and 6.25 l M : P < 0.05) (Fig. 3). Treatment of E ATC with > 50 l M H 2 O 2 resulted in the destruction of the cell population by 24 h, with no change at 25 l M , similarly to baseline (results not shown). 0.28AS-2 cells are less resistant than EATC to H 2 O 2 , as it was found Table 2. Effect of antisense mRNA glutaminase expression on gluta- minase activity and on the intracellular contents of glutamine and glu- tamate. Results represent the mean value ± S D for three different determinations. Assays were perf or med on individual cell culture plates, and the mean of duplicate analysis was used for statistical calculations. Cells Glutaminase (mU per 10 6 cells) Glutamine (m M ) Glutamate (m M ) EATC 23.0 ± 2.8 1.3 ± 0.5 6.7 ± 1.0 0.28AS-2 5.0 ± 0.9* 5.2 ± 1.7* 6.9 ± 1.1 *P< 0.01 comparing control Ehrlich ascitic tumour cell (EATC) values with those of 0.28AS-2 cells. Table 3. Effect of antisense mRNA glutaminase e xpression on the apoptosis lev el determined by fluorescence-activated cell sorter ( FACS) anal ysis, a nd caspase-3 activity, and on glutathione reductase (GR) activity. Results represent the mean values ± SD for three different determinations. Ehrlich ascitic tumour cells (EATC) and 0.28AS-2 cells, untreated or treated for 48 h with 100 n M methotrexate ( MTX) were used for experiments. Experimental conditions are as described in the Materials and methods. Cells Annexin V/FITC + cells (%) Caspase-3 (lmolÆmg )1 protein) GR (unitsÆmg )1 protein) –MTX +MTX –MTX +MTX –MTX +MTX EATC 4.1 ± 0.5 4.2 ± 0.4 139 ± 12 329 ± 31 0.34 ± 0.05 0.16 ± 0.03 0.28AS-2 8.0 ± 0.7* 14.0 ± 0.9* 214 ± 27* 397 ± 18* 0.20 ± 0.02* 0.14 ± 0.02 *P< 0.01 comparing the values of control EATC with those of 0.28AS-2 cells. Ó FEBS 2004 Effect of glutaminase on glutathione and apoptosis ( Eur. J. Biochem. 271) 4301 that incubation of 0.28AS-2 cells for 24 h with 25 l M H 2 O 2 resulted in a similar effect to that seen in EATC incubated with 50 l M H 2 O 2 during the same time-period (results not shown). T hese findings strongly agree with the sensitization to drugs of the cell line expressing antisense GA. After 72 h of treat ment with H 2 O 2 , the growth of EATC and 0.28AS-2 cells was reduced in a time-dependent manner, y ielding I C 50 values of 2 3 and 22 l M , respectively. Although 0.28AS-2 cells showed a greater sensitivity to H 2 O 2 than EATC (Fig. 3), the MTX effect was even greater (Fig. 2 ). In fact, 72 h after the addition of MTX, EATC and 0.28AS-2cellsshowedareducedcellgrowthalsoinatime- dependent manner, with IC 50 values of 52 and 12 n M , respectively. It must also be emphasized that the basal levels of absorban ce at 570 n m (time zero in the absence of a ny drug treatment) for EATC were twice as high as the values for 0.28AS-2 cells (Fig. 1 ). After t reatment of both c ell lines with 100 n M MTX or 100 l M H 2 O 2 , neither were able to grow after the removal of MTX or H 2 O 2 . Apoptosis in response to MTX and H 2 O 2 MTX-induced apoptosis was first demonstr ated in the nucleus by DNA fragmentation, which is a biochemical hallmark o f a poptosis. F igure 4 shows typical DNA fragmentation in cells undergoing apoptosis induced by 100 n M MTX, indicating that this antineoplastic molecule also causes cell death by apoptosis. Similar DNA frag- mentation w as observed i n cells treated with H 2 O 2 ,a chemical that at the assayed concentration has been reported to trigger both morphological change and intra- nucleosomal DNA fragmentation, indicative of apoptosis in many cell types [32]. Not surprisingly, c onsidering the higher toxicity of MTX, this compound induced DNA fragmentation at lower concentrations than seen for H 2 O 2 . In any c ase, DNA fragmentation took pla ce, giving remarkable DNA degradation after exposure of EATC control cells and 0.28AS-2 cells to both 100 n M MTX and 100 l M H 2 O 2 (Fig. 4 ). Fig. 1. Growth curve of Ehrlich ascitic tumour cells (EATC) and 0.28AS-2 cells. Cells were see ded at a c oncentration of 2 · 10 4 mL )1 in a 96-well culture plate. After 24, 48, 72 and 96 h , 3-[4,5-dimethyl- thiazol-2-yl]-2,5-diphenyltetrazoliumbromide(MTT)wasaddedata concentration of 0.5 mg ÆmL )1 and inc ubated f or 3 h, as detailed i n t he Materials and met hods. After 3 0 min, the absorbance of t he sampl e was measured at 570 nm, and the results are shown in the figure. The results represent the mean values f or at least three different wells and are representative of at least three individual experiments, with an SD value of < 10%. Fig. 2. Effect of methotrexate (MTX) on the viability of Ehrlich ascitic tumour cells (EATC) and 0 .28AS-2 c ells. Cells were seeded at a con- centration of 2 · 10 4 mL )1 in a 96-well culture plate, and incubated for 96 h. Then, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro- mide(MTT)wasaddedataconcentrationof0.5mgÆmL )1 and incu- bated for 3 h, as detailed in the Materials and methods. After 30 m in, the absorbance o f t he samp le was measured a t 5 70 nm, and the results are depicted in the figure . Data were normalized to 100% of the untreated control. The results represent the mean values for at least three different wells an d are rep resent ative of at least three in dividual experiments, with an SD of < 10%. Fig. 3. Effect of H 2 O 2 on the viability of Ehrlich ascitic tumour cells (EATC) and 0.28AS-2 cells. Cells were seeded at a co ncentration of 2 · 10 4 mL )1 in a 96-well culture plate, and incubated for 96 h. Then, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added at a conc entratio n of 0 .5 mgÆmL )1 and incubated fo r 3 h, as detailed in the M aterials and methods section. After 30 min, the absorbance of the sample was measured at 57 0 nm, and the results a re depicted in the fi gure. Data are nor malized to 100% o f the untreated control. Re sults represent the mean values for at least three d ifferent wells and are representative of at least three individual experiments, with an SD of < 10%. 4302 J. Lora et al. (Eur. J. Biochem. 271) Ó FEBS 2004 To support th e DNA fragmentation assays, we used annexin V-binding assays to detect the l oss of phospholipid plasma membrane asymmetry and exposure of phosphat- idylserine at the cell s urface, which is an early eve nt in the sequence of e vents that leads to apoptotic cell death. This experiment shows that 48 h of treatment with 100 n M MTX does not affect the r elative number of apo ptotic cells in EATC, but enh ances the ratio of apoptosis (P < 0.01) in 0.28AS-2 cells (Table 3). When comparing EATC and 0.28AS-2 cells in the absence of MTX, twice as many apoptotic cells were found. This effect was found to be augmented at 100 n M MTX, when fourfold more apoptotic 0.28AS-2 c ells were found than control EATC cells (Table 3). Additionally, basal levels (at time zero and in the absence of any added chemical) of MTT staining in 0.28AS-2 cells were found to be lower than those of control EATC (results not shown ). A s observed, EATC cells are more resistant to MTX-induced apoptosis than 0.28AS-2 cells (Fig. 2). On the other h and, antisense GA expression induces apoptosis and sensitizes Ehrlich t umour cells to MTX action. In this study we have confirmed (by assaying the activation of caspase-3, one of the caspase effector s) that MTX induces apoptosis [33]. As shown in Table 3, caspase- 3 activity in both cell lines – control EATC a nd 0.28AS-2 – treatedfor48hwith100n M MTX is highly increased. Caspase-3 activity is augmented, in the presence of MTX, by 2.4-fold and 1.9-fold in EATC and 0 .28AS-2 cells, respectively (Table 3). Of interest, these findings are in agreement with results from the DNA fragmentation and annexin V-FITC a ssays. Discussion The results presented a bove expand on previous work exploring the redox imbalance, consequent to GSH deple- tion, and t he possibility of a relationship b etween apoptosis and tumour proliferation [34]. This work is consistent with others providing evidence of a ntisense technology i nducing apoptosis [35], involving GSH depletion [36], and sensitizing cancer cells to chemotherapy [37]. I n several cases, the findings i n this w ork set the stage for detailed future investigation into various aspects of the role of glutamine and GSH in apoptosis and its implication in selective cancer therapy. The reduced GSH l evels found in 0.28AS-2 cells could be caused by energy depletion and/or a reduced availability of its precursor metabolites as glutamate. Inhibition of GA activity should not result in a straightforward energy depletion, because g lucose is present in the m edium and this metabolite is preferred as an energy substrate by EATC [38]. Consequently, energy status alone might not explain the difference i n GSH le vels. In s pite of simil ar intracellular glutamate concentrations, 0 .28AS-2 cells have significantly higher glutamine levels, reflecting the strong inhibition of GA expression (Table 2). The diminished mitochondrial glutamine catabolism in 0.28AS-2 cells is consistent with the lower GSH levels found in these cells: a positive c orrelation has been found between glutamine supplementation and enhanced production of both intracellular ROS and GSH levels [11]. Moreover, b locking glutamine metabolism by inh ibiting GA causes a significant decrease in ROS production [39]. We reported previously that there exists a mutual dependence b etween glutamine metabolism a nd oxidative stress [4]. c-Glutamylc ysteine is the limiting substrate in the synthesis of GSH. Additionally, c-glut- amylcysteine synthethase, the enzyme that catalyzes the formation of the substrate c-glutamylcysteine, is the rate- limiting enzyme fo r GSH synthesis and i s f eedback inhibited by the production of GSH itself [40]. We reasoned that if an attenuated oxidative metabolism of glutamine is active in these cells, there would b e a lower requirement for GSH as an oxidative scavenger in 0.28AS-2 cells, and a putatively lower c-glutamylcysteine content w ould account for their reduced levels of GSH. In previou s work we demonstrated that epithelial mucin-1 (MUC1) was markedly d iminished in 0.28AS-2 c ells. MUC1 shedding has a protective function agains t the humoral immune response developed against the tumour, so 0.28AS-2 cells were more susceptible to t he immune system response than EATC [41]. It has been reported that MUC1 expression is up-regulated by oxidative stress, demonstra- ting that MUC1 expression is associated with the attenu- ation of ROS levels. It has also been shown that the apoptotic response to o xidative stress is attenuated by a MUC1-dependent mechanism. These r esults suggest a model in which activation of MUC1 by oxidative stress provides a protective function against increased intracellular oxidant levels and ROS-induced apoptosis [42]. Therefore, depleted MUC1 expression of 0.28AS-2 cells agrees with our model, which proposes an inhibition of GSH-dependent antioxidant defence as well as an activation of apoptosis in cells with decreased GA expression. Very re cently, it has b een proposed that glutamine may be protective to cells during periods of oxidative stress, increasing survival in some cell lines through an up-regulation of GSH levels [12]. In addition, the thiol/ disulfide redox state in the intestinal epithelium is an Fig. 4. Agarose gels s howing DNA fragmentation. Cells undergo apoptosis following exposure to 100 l M H 2 O 2 and 100 n M metho- trexate (MTX). Results are representative of three in dividual e xp eri- ments. Control lanes (Ctl) r epresent DNA extracted from untreated Ehrlich ascitic t umour cells (EATC) a nd 0.28AS-2 cells, respectively. H 2 O 2 lanes represent DNA extracted from H 2 O 2 -treated (24 h) EATC and 0.28AS-2 cells, respectively. MTX lanes represent DNA extracted from methotrexate-treated (48 h) EATC and 0.28AS-2 cells, respectively. Ó FEBS 2004 Effect of glutaminase on glutathione and apoptosis ( Eur. J. Biochem. 271) 4303 important determinant of Caco-2 cell proliferation induced by glutamine, enhancing the capability of Caco-2 cells to modulate extremes of extracellular redox [43]. Experimental animal studies have shown that the administration of glutamine increases tissue concentra- tions of reduced GSH. Conversely, glutamine deficiency leads to a cell cycle arrest in G 0 /G 1 and reduces apoptosis. Interestingly, many of these biological activities are also associated with the cellular reduced oxygen potential, which depends mainly on the ratio of reduced GSH to GSSG [44]. Therefore, GSH metabolism is closely related to apoptotic processes. Whether the down-regulation of GA expression and GSH concentrations sensitizes cells to apoptosis only in G 0 /G 1 , or when passing the G 2 /M checkpoint, remains to be investigated. On the other hand, it has been stated previously that administration of glutamine to animals receiving MTX therapy f avours host tolerance to the d rug a nd increases its tumoricidal effectiveness. This effect of MTX i s suggested to be related to GSH me tabolism [45]. Our r eport is strongly in accordance with others, published very recently, proposing t hat lowering the GSH concentration m ay contribute to induce apoptosis in tumour cells [23], i ndicating that systemic oxidative stress, as measured by a d ecrease in GSH levels, is associated with a higher ratio of apoptosis [46] and important redox alterations [47]. These authors have previously demonstra- ted t hat the inhibition of active GSH e xtrusion rescues cells from apoptosis [48]. In contrast, CD95-mediated hepatocyte apoptosis requires an intact intracellular GSH status [49]. The issue of G SH depletion a nd apoptosis has b een a m atter of discussion until the publication of recent articles which indicate that mitochondrial GSH depletion, and not cyto- solic GSH depletion, are critical factors leading to apoptotic tumor cell death activation [50]. In previous work it h as been s hown that a ntisense oligonucleotides, down-regulating the expression of bcl-2 or bcl-xL, induce apoptosis and s ynergistically interact with chemotherapy [51]. Our results, taken together with those discussed above, can be exploited as research tools to gain new insights into the underlying biological basis of the connection of antiproliferative activity of specific antisense GA expression to GSH status a nd apoptosis. I n fact, resistance to chemotherapy has been strongly linked to apoptotic processes [52], and the use of antisense oligo- nucleotides has already provided a rational and promising approach for h elping to overcome chemoresistance i n several malignancies [53]. Because the GA antisense approach has the potential to facilitate apoptosis, using this technique, in c ombination with others, could provide a valuable tool in t herapy. The development o f cancer therapy is an art of integrated sciences, but one of the main points is targeting of genes crucial for cancer cell proliferation. In this field, some interesting results have been achieved very recently using liposome-mediated in vivo gene transfe r and antisense technology [54]. Therefore, GA inhibition and glutamine supplementation deserve further evaluation as potential selective anticancer agents, alone or in combina- tion with cytotoxic drugs in human carcinomas expressing functional GA. However, although similar systems have achieved promising r esults in different human cancer cell lines [55], and even in patients suffering from malignant melanoma [56], further understanding of the clinical usefulness of the proposed combination regimen is required. Acknowledgements We thank E . Manzanares and A. Rubio for valuable help. 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Antisense glutaminase inhibition decreases glutathione antioxidant capacity and increases apoptosis in Ehrlich ascitic tumour cells Jorge Lora, Francisco J. Alonso, Juan A. Segura, Carolina. normal cells. In the present work, Ehrlich ascites tumour cells, and their derivative, 0.28AS-2 cells, expressing antisense glutaminase mRNA, were assayed for apoptosis induced by methotrexate and hydrogen. mour cells, expressing antisense mRNA for glutaminase, contain lower levels of glutathio ne than normal ascites cells. In addition, 0.28AS-2 cells contain a higher number of apop- totic cells and